Calibrator for calibrating time-of-flight mass spectrometry, and preparation method and application thereof

Abstract

The invention relates to the technical field of biology, in particular to a calibrator for calibrating a time-of-flight mass spectrum as well as a preparation method and application of the calibrator. The prepared calibrator is good in stability and can be stably stored at 2-8 DEG C for a long time. When the calibration product is used for calibration, the preparation and use processes are effectively simplified, the storage condition is improved, the pretreatment time is saved, the dependence on personnel and equipment is reduced, the production cost and the use cost are greatly reduced, and the calibration product has a good application prospect and is suitable for large-scale popularization and application.

Description

Technical Field

[0001] The present invention relates to the field of biological detection technologies, and in particular to a calibrator for calibrating a time-of-flight mass spectrometer, a preparation method thereof, and an application thereof. Background Art

[0002] Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (abbreviated as MALDI-TOF MS) is a new type of soft ionization mass spectrometry technology that has rapidly developed since the 1980s of the 20th century and has gradually become popular in Western clinical microbiology laboratories. Thanks to the rapid development of mass spectrometry technology in microbial identification, domestic research scholars have gradually paid attention to the application of MALDI-TOF MS in the field of microbiology. It has the advantages of being fast, accurate, and sensitive in the detection and identification of pathogenic microorganisms. Its principle is to mix a trace amount of sample with an excessive amount of small molecule matrix, spot it on a trace sample target, and after the solvent volatilizes, the matrix and the sample form a co-crystal. After being bombarded by a laser, the matrix absorbs the energy of the laser and is excited, resulting in the ionization and gasification of the sample. Under the action of an electric field, the ionized sample enters the mass analyzer (time-of-flight tube). According to the different mass-to-charge ratios, the time for the sample to reach the detector is different, so that the sample is separated to obtain a corresponding mass spectrum diagram. By comparing with the standard spectrum diagram in the database, the identification of the sample is realized. Among microbial cells, ribosomal proteins are conserved proteins in the cells and have species specificity. The molecular weights of ribosomal proteins are mainly between 2000 and 20000 Da. This is the basis for using a microbial mass spectrometry instrument to identify microorganisms. The instrument is used to collect the spectrum diagram between 2000 and 20000 Da of microbial cells and compare it with the standard database to obtain the name of the best-matched bacterial species. Mass accuracy is an important performance index affecting the accuracy of mass spectrometry microbial identification. It refers to one millionth of the ratio of the difference between the theoretical value and the measured value of a certain protein peak in the standard library to the theoretical value, and is expressed in ppm. Therefore, before using the instrument to identify microorganisms, it is an important prerequisite to use a calibrator to calibrate the instrument to ensure the mass accuracy of the instrument.

[0003] The calibrator for a microbial mass spectrometry instrument is used to calibrate a fully automatic microbial mass spectrometry identification system. According to the calibration principle and the instrument operation instructions, the instrument calibration of the fully automatic microbial mass spectrometry detection system needs to satisfy the time-mass equation: M = f(T, a, b, c), where T is time, and a, b, and c represent coordinate parameters. Theoretically, 3 peaks can satisfy the time-mass equation to achieve instrument calibration. As the number of calibration peaks increases, the calibration stability can be increased. Currently, the main component of the mass calibrator for a mass spectrometer is the protein fingerprint map of the extract of Escherichia coli DH5α strain.

[0004] The technical method adopted by the currently marketed similar calibration products is to prepare a protein mixture, which is sub-packed and then freeze-dried and sub-packed. The calibration-use protein contained in this type of calibration product has a molecular weight evenly distributed between 2000 and 20000 Da. Before performing microbial identification, the instrument is used to collect the spectrum of the calibration product in the range of 2000 - 20000 Da. By adjusting the deviation between the measured value and the standard value of the relevant protein of the instrument parameters, the mass accuracy is controlled within the required range, and the mass drift of the instrument is reduced.

[0005] The technical method adopted by the currently marketed similar calibration products is to prepare a protein mixture, which is sub-packed, freeze-dried and then packaged. Not only is the preparation process complex (requiring freeze-drying, usually taking more than 6 hours), the preparation cost is high, but also the storage conditions are demanding (stored at -20°C). When users perform mass spectrometry instrument calibration, they also need to perform the following operations:

[0006] 1. Take 15 μL of lysis solution 1 (mass spectrometry sample pretreatment reagent), add it to the calibration product reagent tube, and pipette about 5 times. Try to avoid generating bubbles during the pipetting process.

[0007] 2. Add 15 μL of lysis solution 2 (mass spectrometry sample pretreatment reagent), and pipette about 5 times.

[0008] 3. Centrifuge at 13000 rpm for 2 min.

[0009] 4. Pipette 1 μL of the supernatant and spot it on the sample target, and let it dry.

[0010] 5. Pipette 1 μL of the matrix solution (mass spectrometry sample pretreatment reagent) and cover the above sample spot, and let it dry.

[0011] 6. Spot the test sample on other sample target spots.

[0012] 7. Place the target in the mass spectrometer and operate it according to the "Operation Manual of the Automatic Microbial Mass Spectrometry Detection System".

[0013] The entire operation process before the calibration product is put into the instrument takes about 5 minutes, requires the cooperation of mass spectrometry sample pretreatment reagents, and requires test instruments such as a centrifuge. Not only are there many operation steps, but also certain test operation skills and personnel requirements are needed, and the requirements for the instrument and supporting reagent kits are also high. Summary of the Invention

[0014] In view of this, the present invention provides a calibration product for calibrating time-of-flight mass spectrometry, its preparation method and application. The preparation process of this calibration product is simple, takes a short time, the calibration process is simple and fast, has low requirements for storage conditions, and has high stability.

[0015] In order to achieve the above-mentioned invention purpose, the present invention provides the following technical solutions:

[0016] Preparation method of calibration product, comprising:

[0017] Step 1: Mix purified water, acetonitrile, absolute ethanol and trifluoroacetic acid to obtain a standard solvent;

[0018] Step 2: Mix α-cyano-4-hydroxycinnamic acid and the standard solvent described in Step 1 to obtain a matrix solution;

[0019] Step 3: Mix the Escherichia coli extract and the standard solvent described in Step 1, let it stand, and then add the matrix solution to obtain a calibration product.

[0020] In the present invention, a calibration product is prepared by mixing purified water, acetonitrile, absolute ethanol, trifluoroacetic acid, DH5α strain extract, and matrix in a certain order. Experiments show that the calibration product has good stability, can be stored stably at 2-8°C for a long time, and does not require the freezing condition of -20°C for traditional calibration products. In addition, the calibration product does not need to be freeze-dried, has a short preparation time, and a low preparation cost.

[0021] The calibration product of the present invention can be directly dropped onto the target plate. After natural drying, it can be directly loaded onto the automatic microbial mass spectrometry identification system for calibration, without the need for the pretreatment operations of traditional calibration products (such as centrifugation, adding other reagents, etc.). The sample addition time for a single calibration product is less than 10 seconds, which effectively simplifies the operation process, saves the pretreatment time, reduces the dependence on personnel and equipment, and the requirements for storage conditions, and greatly reduces the use cost.

[0022] In the present invention, in the standard solvent described in Step 1, the volume ratio of the purified water, acetonitrile, absolute ethanol and trifluoroacetic acid is (42.5-50.5):(45-51):(2.5-4.5):(2-4). In some specific embodiments, the volume ratio is specifically: 45.5:48:3.5:3, 50.5:45:2.5:2, 48.5:45:3.5:3, 46.5:45:4.5:4, 46.5:48:2.5:3, 44.5:48:3.5:4, 45.5:48:4.5:2, 42.5:51:2.5:4, 42.5:51:3.5:3, 42.5:51:4.5:2. Among them, the calibration product solution prepared when the volume ratio is 45.5:48:3.5:3 has the best stability.

[0023] In the present invention, in the matrix solution described in Step 2, the concentration of α-cyano-4-hydroxycinnamic acid is 8-12 mg / ml, and specifically can be 8 mg / ml, 9 mg / ml, 10 mg / ml, 11 mg / ml, 12 mg / ml.

[0024] Experiments show that the calibration standards prepared by preparing standard solutions and matrix solutions according to the above ratios and concentrations can all be automatically calibrated to standards by mass spectrometers after being stored at 2-8°C for 12 months.

[0025] In step 2 of the present invention, the mixing is carried out under stirring conditions, and the stirring speed is 200-800 r / min, preferably 400-600 r / min, and specifically can be 400 r / min, 500 r / min, 600 r / min. In the present invention, the stirring time ≥15 min, and the specific time can be reasonably selected according to the actual situation.

[0026] In step 3 of the present invention, in every 100-400 μl of the calibration standard, the dosage of the Escherichia coli extract is 5-20 mg, preferably 8-12 mg, and specifically can be 5 mg, 8 mg, 10 mg, 12 mg.

[0027] In step 3 of the present invention, the volume ratio of the standard solvent to the matrix solution is 100:(100-300).

[0028] In step 3 of the present invention, after standing, it further includes a filtration step. The filtration includes filtration using a filter membrane. Further, the pore size of the filter membrane for filtration includes 0.22 μm. In a specific embodiment of the present invention, the filtration is: filtration is carried out using a 0.22-μm filter membrane.

[0029] In the present invention, the Escherichia coli extract can be directly purchased commercially or prepared by oneself. In a specific embodiment of the present invention, the Escherichia coli extract is an extract of DH5α strain, purchased from Zhengzhou Yimeinuo Biotechnology Co., Ltd.; product number: AA1930101.

[0030] The present invention also provides a calibration standard prepared by the above preparation method.

[0031] After mass spectrometry detection, the calibration standard of the present invention contains 7 calibration peaks, and the mass-to-charge ratios of the calibration peaks include 3637.772 Da, 4365.343 Da, 5096.776 Da, 5381.446 Da, 6255.444 Da, 7274.467 Da, 10300.032 Da.

[0032] The present invention also provides a kit for microbial mass spectrometry identification, which includes the calibration standard of the present invention.

[0033] The present invention also provides the application of the calibration standard or the kit in calibrating time-of-flight mass spectrometry detection.

[0034] The present invention also provides a method for using the calibration standard. Pipette 1 μl of the calibration standard prepared by the present invention onto the target plate, and it can be calibrated on the machine after drying.

[0035] Alternatively, aspirate 1 μl of the calibrator prepared by the present invention onto the target plate. After it dries and is evacuated, store it at 2 - 8°C for one year. When it is needed, open the package and load it onto the instrument.

[0036] The present invention prepares a calibrator by mixing purified water, acetonitrile, absolute ethanol, trifluoroacetic acid, DH5α strain extract, and matrix in a certain order and proportion. The calibrator prepared by the present invention has good stability and can be stably stored at 2 - 8°C for a long time. Using this calibrator for calibration effectively simplifies the preparation, usage process, and storage conditions, saves the time for pretreatment, has low dependence on personnel and equipment, greatly reduces the production cost and usage cost, has good application prospects, and is suitable for large-scale popularization and application. Description of the Drawings

[0037] Figure 1 Calibration peak chart of the calibrator prepared in Example 1 stored for 1 month;

[0038] Figure 2 Calibration peak chart of the calibrator prepared in Example 1 stored for 3 months;

[0039] Figure 3 Calibration peak chart of the calibrator prepared in Example 1 stored for 6 months;

[0040] Figure 4 Calibration peak chart of the calibrator prepared in Example 1 stored for 9 months;

[0041] Figure 5 Calibration peak chart of the calibrator prepared in Example 1 stored for 12 months;

[0042] Figure 6 Calibration peak chart of the calibrator prepared with Test Ratio 1 in Test Example 2;

[0043] Figure 7 Calibration peak chart of the calibrator prepared with Test Ratio 2 in Test Example 2;

[0044] Figure 8 Calibration peak chart of the calibrator prepared with Test Ratio 3 in Test Example 2;

[0045] Figure 9 Calibration peak chart of the calibrator prepared with Test Ratio 4 in Test Example 2;

[0046] Figure 10 Calibration peak chart of the calibrator prepared with Test Ratio 5 in Test Example 2;

[0047] Figure 11 Calibration peak chart of the calibrator prepared with Test Ratio 6 in Test Example 2;

[0048] Figure 12Calibration peak map of the calibrator prepared with test ratio 7 in Test Example 2;

[0049] Figure 13 Calibration peak map of the calibrator prepared with test ratio 8 in Test Example 2;

[0050] Figure 14 Calibration peak map of the calibrator prepared with test ratio 9 in Test Example 2;

[0051] Figure 15 Calibration peak map of the calibrator after the calibrator of Example 1 was spotted on the target plate and stored in vacuum for 6 months in Test Example 3;

[0052] Figure 16 Calibration peak map of the calibrator after the calibrator of Example 1 was spotted on the target plate and stored in vacuum for 12 months in Test Example 3;

[0053] Figure 17 Calibration peak map of Control Group 1 in Test Example 4;

[0054] Figure 18 Calibration peak map of Control Group 2 in Test Example 4;

[0055] Figure 19 Calibration peak map of the experimental group in Test Example 4. Detailed implementation manners

[0056] The present invention provides a calibrator for mass spectrometry identification of Escherichia coli, its preparation method and application. Those skilled in the art can draw on the content of this article and appropriately improve the process parameters to achieve. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art, and they are all regarded as included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and those skilled in the art can obviously make changes or appropriate changes and combinations to the methods and applications herein without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0057] In the present invention, the calibrator refers to a purified protein, which is used to calibrate and eliminate the influence on the detection result caused by the fluctuation of the operating conditions or the instrument state, and avoid the deviation of the detection result caused by drift.

[0058] In the present invention, in the matrix solution, the matrix (HCCA) is α-cyano-4-hydroxycinnamic acid, which is used for mass spectrometry identification of clinical microorganisms. When in use, the matrix can absorb laser energy and transfer the energy to microbial proteins to cause ionization to generate charged particles. The particles pass through the flight system and are finally detected by the AUTOF MS detector to generate a current signal. After software processing, a mass spectrometry map presented in the form of the protein charge ratio can be generated.

[0059] The test materials used in the present invention are all ordinary commercially available products and can be purchased on the market.

[0060] The present invention will be further described below in conjunction with embodiments:

[0061] Example 1

[0062] 1. Preparation of standard solvent:

[0063] Add purified water, acetonitrile, absolute ethanol, and trifluoroacetic acid into a container, stir for ≥3 minutes until completely mixed evenly, and it is the standard solvent.

[0064] Table 1

[0065] Name 100 mL standard volume Purified water 45.5 mL Acetonitrile 48 mL Absolute ethanol 3.5 mL Trifluoroacetic acid 3.0 mL

[0066] 2. Preparation of matrix solution:

[0067] Add α-cyano-4-hydroxycinnamic acid into the above-mentioned standard solvent, stir thoroughly to dissolve, stirring requirements: 200 - 800 revolutions or medium speed, stirring time ≥15 min, to obtain the matrix solution.

[0068] Table 2

[0069] Name 100 mL standard volume Standard solvent 100ml α-Cyano-4-hydroxycinnamic 1g

[0070] 3. Restore the DH5α strain extract to room temperature, add the standard solvent, pipette and blow 5 - 6 times, let it stand for 2 min, then add the matrix solution, pipette and blow 5 - 6 times, and let it stand for 2 min. Filter using a 0.22 μm PES filter membrane. After filtration, the calibrator solution should be clear and transparent without visible particles to the naked eye. After aliquoting, store at 2 - 8 °C.

[0071] Table 3

[0072] Name 30 mL standard volume DH5α strain extract 1000 mg Standard solvent 10 mL Matrix solution 20 mL

[0073] 4. Usage method: Pipette 1 μl of the calibrator solution obtained in step 3 onto the target plate, and wait for it to dry before calibrating on the machine.

[0074] The calibrator prepared in this example contains 7 calibration peaks (3637.772 Da, 4365.343 Da, 5096.776 Da, 5381.446 Da, 6255.444 Da, 7274.467 Da, 10300.032 Da).

[0075] According to the calibration principle: Instrument calibration needs to satisfy the time-quality equation: M = f(T, a, b, c), where T is time, and a, b, and c represent calibration parameters, representing three different calibration peaks respectively. Theoretically, 3 peaks can satisfy the time-quality equation to achieve instrument calibration. However, a fourth peak is required to verify the correctness of the time-quality equation. Therefore, there are at least 4 calibration peaks, and instrument calibration can also be achieved when the error < 300 ppm. As the number of calibration peaks increases, the sample size is increased, which can improve the stability of calibration, but also increases the cost. The calibration product of the present invention contains 7 calibration peaks, effectively increasing the sample size, and greatly reducing both the preparation cost and the usage cost, having obvious advantages compared with traditional calibration products.

[0076] Test Example 1

[0077] Prepare the calibration product solution according to Example 1, and after storing it at 2 - 8°C, use it for mass spectrometer calibration respectively. Mass spectrometer: Antu Biotech - Automatic Microbial Mass Spectrometry Detection System Autofms series.

[0078] Calibration parameters: Error 500 ppm, 7 calibration peaks (3637.772 Da, 4365.343 Da, 5096.776 Da, 5381.446 Da, 6255.444 Da, 7274.467 Da, 10300.032 Da).

[0079] The storage times are 1 month, 3 months, 6 months, 9 months, and 12 months respectively. The results are shown in Figures 1 - 5 .

[0080] The results show that after being stored for 1 - 12 months, the calibration product of Example 1 can all pass the automatic calibration of the mass spectrometer as the standard, and there are no obvious changes in the peak pattern and peak intensity. It shows that the calibration product provided by the present invention has a stable calibration effect.

[0081] Test Example 2

[0082] Test the calibration effects of calibration products with different ratios.

[0083] Prepare the standard solvent and matrix solution according to the method of Example 1, and prepare calibration product solutions with different ratios according to the ratios in Table 4. The dosage of each 200 - 400 μl calibration product solution and the test ratios are shown in Table 4. The results are shown in Figures 6 - 14 .

[0084] Table 4

[0085]

[0086] The results show that the calibration product solutions prepared with test ratios 1 - 9 can also pass the calibration. Among them, the peak intensity of ratio 5 reaches 8000, and the baseline is significantly lower than 500, with the best effect.

[0087] Test Example 3

[0088] Add the calibrator prepared in Example 1 to the target plate and store it under vacuum. Detect the calibration effect after different storage times (6 months, 12 months), as shown in Figures 15 - 16 .

[0089] Conclusion: Add the calibrator prepared in Example 1 to the target plate and store it under vacuum at 2-8°C for 12 months, and it can still be automatically calibrated by the mass spectrometer.

[0090] Test Example 4

[0091] During the preparation of the test calibrator, investigate the influence of the mixing order of the strain extract, standard solvent and matrix solution on the calibration effect:

[0092] Prepare the standard solvent and matrix solution according to Example 1. Adjust the mixing order of the DH5α strain extract, standard solvent and matrix solution. Prepare the calibrator solution according to the ratio 5 in Table 4 of Test Example 2, and set the following control groups 1-2 and experimental group:

[0093] Control Group 1: First mix the strain extract with the matrix solution, then add it to the standard solvent. Other steps and parameters are the same as in Example 1, and test the calibration effect; the results are shown in Figure 17 .

[0094] Control Group 2: Mix the matrix solution with the standard solvent, then add the strain extract. Other steps and parameters are the same as in Example 1, and test the calibration effect. The results are shown in Figure 18 .

[0095] Experimental Group: According to the method of Example 1 of the present invention, mix the standard solvent with the strain extract, and then add the matrix solution, and test the calibration effect. The results are shown in Figure 19 .

[0096] Conclusion: Compared with Control Groups 1-2, the calibrator prepared according to the method of the present invention can make the combination of the matrix and protein more compact, with the best peak map quality, which is more conducive to the calibration of the mass spectrometer.

[0097] The above is only the preferred embodiment of the present invention. It should be pointed out that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. A method for preparing a calibrator, characterized in that: include: Step 1, mixing purified water, acetonitrile, anhydrous ethanol and trifluoroacetic acid to obtain a standard solvent; Step 2, mixing α-cyano-4-hydroxycinnamic acid and the standard solvent described in step 1 to obtain a matrix solution; Step 3: Mix the Escherichia coli extract and the standard solvent described in step 1, let stand, and then add the matrix solution to obtain a calibrant.

2. The preparation method according to claim 1, characterized in that: In step 1, in the standard solvent, the volume ratio of purified water, acetonitrile, anhydrous ethanol and trifluoroacetic acid is (42.5-50.5):(45-51):(2.5-4.5):(2-4).

3. The preparation method according to claim 1 or 2, characterized in that: The volume ratio of the purified water, acetonitrile, anhydrous ethanol and trifluoroacetic acid is 45.5:48:3.5:

3.

4. The preparation method according to claim 1, characterized in that: In step 2, the concentration of α-cyano-4-hydroxycinnamic acid in the matrix solution is 8 to 12 mg / ml.

5. The preparation method according to claim 4, characterized in that: In the matrix solution, the concentration of α-cyano-4-hydroxycinnamic acid is 10 mg / ml.

6. The preparation method according to claim 1, characterized in that: In step 2, the mixing is carried out under stirring conditions, the stirring speed is 200-800r / min, and the time is ≥15min.

7. The preparation method according to claim 1, wherein in step 3, the amount of the Escherichia coli extract in every 200 to 400 μl of the calibrator is 5 to 20 mg.

8. The preparation method according to claim 1, in step 3, the volume ratio of the standard solvent to the matrix solution is 100:(100-300).

9. The preparation method according to claim 1, characterized in that: In step 3, after standing, the step of filtering using a filter membrane is also included; the pore size of the filter membrane is 0.22 μm.

10. The preparation method according to any one of claims 1 to 9, characterized in that: The Escherichia coli includes a DH5α strain.

11. A calibrator prepared according to the method of any one of claims 1 to 9.

12. The calibrator according to claim 11, characterized in that It contains 7 calibration peaks, and the mass-to-charge ratios of the calibration peaks include 3637.772 Da, 4365.343 Da, 5096.776 Da, 5381.446 Da, 6255.444 Da, 7274.467 Da, and 10300.032 Da.

13. A kit for mass spectrometry identification of Escherichia coli, characterized in that: Including the calibrator prepared by any one of the preparation methods of claims 1 to 10 or the calibrator described in any one of claims 11 to 12.

14. Use of the calibrator prepared by the preparation method of any one of claims 1 to 10, the calibrator according to any one of claims 11 to 12, or the kit according to claim 13 in calibrating time-of-flight mass spectrometry.

Images